Production of multimeric protein by cell fusion method

ABSTRACT

The present invention features a method of producing a multimeric protein from a hybrid cell formed from the fusion of two or more cells, each of which cell is engineered to express one component of the multimeric protein, as well as a method for screening for successful fusion of the cells to produce a desired hybrid cell. The methods of the invention are widely applicable to the production of proteins having two or more components.

FIELD OF THE INVENTION

[0001] This invention relates generally to methods for use in geneexpression and cell fusion techniques, particularly in the production ofmulti-component proteins.

BACKGROUND OF THE INVENTION

[0002] Recombinant DNA techniques have been used for production ofheterologous proteins in transformed host cells. Generally, the producedproteins are composed of a single amino acid chain or two chains cleavedfrom a single polypeptide chain. More recently, multichain proteins suchas antibodies have been produced by transforming a single host cell withDNA sequences encoding each of the polypeptide chains and expressing thepolypeptide chains in the transformed host cell (U.S. Pat. No.4,816,397).

[0003] The basic immunoglobulin (Ig) structural unit in vertebratesystems is composed of two identical “light” polypeptide chains(approximately 23 kDa), and two identical “heavy” chains (approximately53 to 70 kDa). The four chains are joined by disulfide bonds in a “Y”configuration, and the “tail” portions of the two heavy chains are boundby covalent disulfide linkages when the immunoglobulins are generatedeither by hybridomas or by B cells.

[0004] A schematic of the general antibody structure is shown in FIG. 1.The light and heavy chains are each composed of a variable region at theN-terminal end, and a constant region at the C-terminal end. In thelight chain, the variable region (termed “V_(L)J_(L)”) is the product ofthe recombination of a V_(L) gene to a J_(L) gene. In the heavy chain,the variable region (V_(H)D_(H)J_(H)) is the product of recombination offirst a D_(H) and a J_(H) gene, followed by a D_(H)J_(H) to V_(H)recombination. The VLJ_(L) and V_(H)D_(H)J_(H) regions of the light andheavy chains, respectively, are associated at the tips of the Y to formthe antibody's antigen binding domain and together determine antigenbinding specificity.

[0005] The (C_(H)) region defines the antibody's isotype, i.e., itsclass or subclass. Antibodies of different isotypes differ significantlyin their effector functions, such as the ability to activate complement,bind to specific receptors (Fc receptors) present on a wide variety ofcell types, cross mucosal and placental barriers, and form polymers ofthe basic four-chain IgG molecule.

[0006] Antibodies are categorized into “classes” according to the C_(H)type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, orIgA). There are at least five types of C_(H) genes (Cμ, Cγ, Cδ, Cε, andCα), and some species (including humans) have multiple CH subtypes(e.g., Cγ₁, Cγ₂, Cγ₃, and Cγ₄ in humans). There are a total of nineC_(H) genes in the haploid genome of humans, eight in mouse and rat, andseveral fewer in many other species. In contrast, there are normallyonly two types of light chain constant regions (C_(L)), kappa (κ) andlambda (λ), and only one of these constant regions is present in asingle light chain protein (i.e., there is only one possible light chainconstant region for every V_(L)J_(L) produced). Each heavy chain classcan be associated with either of the light chain classes (e.g., a C_(H)γregion can be present in the same antibody as either a κ or λ lightchain).

[0007] A process for the immortalization of B cell clones producingantibodies of a single specificity has been developed involving fusing Bcells from the spleen of an immunized mouse with immortal myeloma cells.Single clones of fused cells secreting the desired antibody could thenbe isolated by drug selection followed by immunoassay. These cells weregiven the name “hybridoma” and their antibody products termed“monoclonal antibodies.”

[0008] The use of monoclonal antibodies as therapeutic agents for humandisease requires the ability to produce large quantities of the desiredantibody. One approach to increased production was simply to scale upthe culture of hybridoma cells. Although this approach is useful, it islimited to production of that antibody originally isolated from themouse. In the case where a hybridoma cell produces a high affinitymonoclonal antibody with the desired biological activity, but has a lowproduction rate, the gene encoding the antibody can be isolated andtransferred to a different cell with a high production rate.

[0009] In some cases it is desirable to retain the specificity of theoriginal monoclonal antibody while altering some of its otherproperties. For example, a problem with using murine antibodies directlyfor human therapy is that antibodies produced in murine systems may berecognized as “foreign” proteins by the human immune system, eliciting aresponse against the antibodies. A human anti-murine antibody (HAMA)response results in antibody neutralization and clearance and/orpotentially serious side-effects associated with the anti-antibodyimmune response. Such murine-derived antibodies thus have limitedtherapeutic value.

[0010] One approach to reducing the immunogenicity of murine antibodiesis to replace the constant domains of the heavy and light chains withthe corresponding human constant domains, thus generating human-murinechimeric antibodies. Chimeric antibodies are generally produced bycloning the antibody variable regions and/or constant regions, combiningthe cloned sequences into a single construct encoding all or a portionof a functional chimeric antibody having the desired variable andconstant regions, introducing the construct into a cell capable ofexpressing antibodies, and selecting cells that stably express thechimeric antibody. Examples of methods using recombinant DNA techniquesto produce chimeric antibodies are described in PCT Publication No. WO86/01533 (Neuberger et al.), and in U.S. Pat. No. 4,816,567 (Cabilly etal.) and U.S. Pat. No. 5,202,238 (Fell et al.).

[0011] In another approach, complementarity determining region(CDR)-grafted humanized antibodies have been constructed bytransplanting the antigen binding site, rather than the entire variabledomain, from a rodent antibody into a human antibody. Transplantation ofthe hypervariable regions of an antigen-specific mouse antibody into ahuman heavy chain gene has been shown to result in an antibody retainingantigen-specificity with greatly reduced immunogenicity in humans(Riechmann et al. (1988) Nature 332:323-327; Caron et al. (1992) J. Exp.Med 176:1191-1195).

[0012] Another approach in the production of human antibodies has beenthe generation of human B cell hybridomas. Applications of human B cellhybridoma-produced monoclonal antibodies have promising potential in thetreatment of cancer, microbial infections, B cell immunodeficienciesassociated with abnormally low antibody production, and other diseasesand disorders of the immune system. Obstacles remain in the developmentof such human monoclonal antibodies. For example, many human tumorantigens may not be immunogenic in humans and thus it may be difficultto isolate anti-tumor antigen antibody-producing human B cells forhybridoma fusion.

[0013] For a given disease indication, one antibody isotype is likely tobe greatly preferred over another. The preferred isotype may vary fromone indication to the next. For example, to treat cancer it may bedesirable that the binding of an antibody to a tumor cell result inkilling of a tumor cell. In this case, an IgG1 antibody, which mediatesboth antibody-dependent cellular cytotoxicity and complement fixation,would be the antibody of choice. Alternatively, for treating anautoimmune disease, it may be important that the antibody only blockbinding of a ligand to a receptor and not cause cell killing. In thiscase, an IgG4 or IgG2 antibody would be preferred. Thus, even in asituation where a high affinity, antigen-specific, fully human antibodyhas been isolated, it may be desirable to re-engineer that antibody andexpress the new product in a different cell.

[0014] The growing use of phage display technology also points to a needfor antibody engineering and expression methodologies. Phage displaytechnology is used for producing libraries of antibody variable domainscloned into bacteria. This allows variable domains of desiredspecificity to be selected and manipulated in vitro. While bacteriaoffer a great advantage for selecting and producing antibody fragments,they are not capable of producing fullsize intact antibodies in nativeconfiguration, and it is necessary to reconstitute fragments selected inbacteria into intact antibodies and express them in eucaryotic cells.

SUMMARY OF THE INVENTION

[0015] The present invention features a method of producing a multimericprotein from a hybrid cell formed from the fusion of two or more cells,each of which cell is engineered to express one component of themultimeric protein, as well as a method for screening for successfulfusion of the cells to produce a desired hybrid cell. The methods of theinvention are widely applicable to the production of proteins having twoor more components.

[0016] In one specific application of the method of the invention, themultimeric protein is an antibody composed of antigen-specific heavy andlight chains. DNA encoding the desired heavy chain (or a fragment of theheavy chain) is introduced into a first mammalian host cell, while DNAencoding the desired light chain (or a fragment of the light chain) isintroduced into a second mammalian host cell. The first transformed hostcell and the second transformed host cell are then combined by cellfusion to form a third cell. Prior to fusion of the first and secondcells, the transformed cells may be selected for specifically desiredcharacteristics, e.g., high levels of expression. After fusion, theresulting hybrid cell contains and expresses both the DNA encoding thedesired heavy chain and the DNA encoding the desired light chain,resulting in production of the multimeric antibody.

[0017] In one aspect the invention features the multimeric proteinproduced by the method of the invention. In one embodiment, theinvention includes an antibody produced by the method of the invention.

[0018] In another aspect the invention features a method for screeningfor successful fusion of a first cell containing a first nucleotidesequence encoding a desired antibody heavy chain and a second cellcontaining a second nucleotide sequence encoding a desired antibodylight chain, the method comprising including a nucleotide sequenceencoding a first marker gene in the first cell, including a nucleotidesequence encoding a second marker gene in the second cell, fusing thefirst and second cells to produce a fused cell and assaying for thepresence of the first and second marker genes in the fused cell.

[0019] One advantage of the method of the invention is that cellsexpressing a single component of the final multi-component protein canbe individually selected for one or more desired characteristics, suchas a high rate of production.

[0020] Another advantage is that the method generates a cell whichproduces an antibody at a multiplication high rate through the fusion oftwo kinds of cells which are each selected prior to fusion for highproduction of the desired heavy or light chains.

[0021] Another advantage is that the final multi-component protein isnot expressed until all the cells expressing the individual componentsof the multi-component protein are fused into a single hybrid cell.

[0022] Other aspects, features, and advantages of the invention willbecome apparent from the following detailed description, and the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1 is a schematic showing the basic immunoglobulin structure.

[0024]FIG. 2 is a flow chart showing one embodiment of the method ofinvention when mammalian cells are separately transformed with thedesired light and heavy chain DNA, then fused to form the hybrid cellexpressing both chains.

[0025]FIG. 3 illustrates a specific embodiment of the invention in whicha mammalian cell expressing an irrelevant light chain is transformedwith the desired heavy chain DNA, a second mammalian cell is transformedwith the desired light chain DNA, and the desired hybrid cell formedfrom fusion of the transformed host cells is selected which expressesthe desired antibody product.

[0026]FIG. 4 is a schematic illustrating a specific embodiment of theinvention in which DHFR CHO cells are independently transfected with (i)pManuGamma#6, a human heavy chain Ig construct and (ii) pManuKappa#14, ahuman light chain Ig construct. The independent cell lines are selected,amplified, fused, and selected to yield a hybrid cell containing thehuman heavy chain Ig construct and the human light chain Ig construct.

[0027]FIG. 5 is a schematic diagram of a fusion method in accordancewith the present invention demonstrating the use of HPRT and LacZ markergenes for the initial determination of the success of a fusion process.

DETAILED DESCRIPTION

[0028] Before the methods and compositions of the present invention aredescribed and disclosed it is to be understood that this invention isnot limited to the particular methods and compositions described as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting since the scope of the presentinvention will be limited only by the appended claims.

[0029] It must be noted that as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, reference to “a DNA sequence” includes a plurality of DNAsequences and different types of DNA sequences.

[0030] Unless defined otherwise all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any materialsor methods similar or equivalent to those described herein can be usedin the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the particular information for which thepublication was cited. The publications discussed above are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventor is not entitled to antedate such disclosure by virtue of priorinvention.

Definitions

[0031] By the term “nucleotide sequence” is meant any DNA fragment ofinterest which may be introduced into a cell, including an intact geneor fragment of a gene. When the method of the invention is used togenerate an antibody, the nucleotide sequence of interest will be all orpart of either the constant region and/or variable region of the lightor heavy chains, and may include all, part, or none of the regulatorynucleotide sequences that control expression of the light or heavychain. The nucleotide sequence of interest for heavy chains includes butis not limited to all or a portion of the V, D, J, and switch regions(including intervening sequences) and flanking sequences. For lightchains, the nucleotide sequence of interest includes but is not limitedto the V and J regions, and flanking and intervening sequences. Thenucleotide sequence may be a naturally occurring sequence, synthetic, orpartially natural and partially synthetic. The sequence may also be anon-naturally occurring or modified naturally-occurring sequence. TheDNA sequence includes sequences taken from different sources, e.g.,different species. For example, when the method is used to produce anantibody, the DNA chain may encode a chimeric (for example, human-mouse)immunoglobulin chain, or it may be a CDR-grafted DNA sequence having ahuman immunoglobulin sequence with antigen-specific murine CDRsequences. The DNA of the nucleotide sequence may encode a fully humanantibody. B-cells obtained from non-human animals immunized with anantigen and also hybridoma, trioma, and quadromas derived from suchB-cells can also provide the nucleotide sequence introduced into thehost cells. B-cells and hybridomas producing any kind of monoclonalantibody may be used as a source of the nucleotide sequence, includingcells producing, for example, fully mouse monoclonal antibodies, fullyhuman monoclonal antibodies, CDR-grafted monoclonal antibodies, chimericmonoclonal antibodies, and F(ab)₂.

[0032] By the terms “multi-component”, “multichain”, or “multimeric”protein is meant a protein composed of two or more proteins orpolypeptides. The method of the invention is useful for producing amultimeric protein by the fusion of two or more cells each expressing asingle component of the multimeric protein. For example, in oneembodiment the multi-component protein is an antibody generated from twoheavy chains encoded by DNA transfected into a first cell and two lightchains encoded by DNA transfected into a second cell, where the finalmultimeric antibody is produced by a hybrid cell formed from the fusionof the first and second cells. “Multi-component,” “multichain,” and“multimeric” protein is meant to include any heterodimeric orhetero-oligomeric protein (e.g., BMP2/BMP7 heterodimeric osteogenicprotein, ICE (interleukin-1 converting protein), receptors of thenucleus (e.g., retinoid receptors), heterodimeric cell surface receptors(e.g., T cell receptors), integrins (e.g., cell adhesion molecules,β₁-integrins, (see, e.g., Hynes, 1987 Cell 48:549-554; Hynes 1992 Cell60:11-25), tumor necrosis factor (TNF) receptor, and soluble andmembrane-bound forms of class I and class II MHC (majorhistocompatibility complex proteins). Where the multimeric protein is areceptor, “multimeric protein” is meant to encompass soluble andmembrane forms of the receptor.

[0033] By the term “introducing” a nucleotide sequence into a cell meansinserting an exogenous piece of DNA into a cell, including but notlimited to transfection or transduction with a vector, such that all orpart of the exogenous nucleotide sequence is stably maintained in thecell, and the resulting transformed cell expresses the introducednucleotide sequence.

[0034] By the term “fusing” or “fusion” of two or more cells is meant amethod in which two or more cells are combined to form a single hybridcell which contains all or part of at least the nucleic acid content ofeach individual cell. Fusion may be accomplished by any method ofcombining cells under fuseogenic conditions well known in the art (See,for example, Harlow & Lane (1988) in Antibodies, Cold Spring HarborPress, New York). Known methods for fusing cells includes by use withpolyethylene glycol (PEG) or Sendai virus.

[0035] By the term “hybrid cell” is meant a cell formed by combining twoor more cells, e.g., by fusion. In the method of the invention, hybridcells are formed from the fusion of one or more transformed cells eachexpressing a single component of a multimeric protein.

[0036] The term “irrelevant” as in, e.g., “an irrelevant light chain”means a light chain which does not contribute to the binding of theantigen of interest and is not a component of the multimeric proteinproduced by the hybrid cell of the invention.

[0037] By the term “desired” component, e.g., desired heavy chain, ordesired light chain, is meant an immunoglobulin chain which recognizesthe antigen of interest.

Generation of a Hybrid Cell Producing a Heterologous Multimeric Protein

[0038] The present invention provides a method for generating a hybridcell producing a multi-component protein from two or more transformedcells each of which cells produces a single component of the multimericprotein. This method features several important advantages relative toconventional methods for protein production. For example, the method ofthe present invention allows separately transformed cells to beindividually selected for optimal expression of each component of themulti-component protein. This selection occurs prior to fusion of cellsforming the hybrid cell and prior to production of the final multimericprotein. The method of the invention results in a final multi-componentprotein product which is not expressed until a single hybrid cell isproduced from the fusion of each cell expressing a component of thefinal protein product.

[0039] Generally, when the multi-component protein to be produced is anantibody, the method of the invention involves generation of a cellexpressing a desired heavy chain, generation of a cell expressing adesired light chain, and fusion of the two cells to form a hybrid cellexpressing the final antibody protein (FIG. 2). Generation of a cellexpressing the desired heavy chain involves the following steps: (1)identifying and cloning and/or synthesizing the gene, gene fragment, ornucleotide sequence encoding the variable segment or antigen-bindingsequences of the heavy chain. The nucleotide sequence may be obtainedfrom either a cDNA or genomic source, or synthesized de novo; (2)cloning the nucleotide sequence encoding the desired constant regions ofthe heavy chain; (3) ligating the variable region with the constantregion so that the complete nucleotide sequence can be transcribed andtranslated to express the desired heavy chain polypeptide; (4) ligatingthe construct into a vector containing a selectable marker andappropriate gene control regions; (5) amplifying the construct inbacteria; (6) introducing the vector into eukaryotic cells; (7)selecting the cells expressing the selectable marker; and (8) screeningthe cell supernatants or lysates for the expressed heavy chain.Similarly, a cell expressing a desired light chain construct isgenerated as outlined above.

[0040] Alternatively, the process of generating a cell expressing adesired heavy or light chain may involve (1) construction of a Ig chainDNA sequence containing (a) a signal sequence, (b) the gene, genefragment, or nucleotide sequence encoding the variable region orantigen-binding sequences, and (c) the nucleotide sequence encoding thedesired constant region of the Ig chain, followed by (2) PCRamplification of the Ig construction, (3) insertion of the constructinto eukaryotic cells, (4) selecting the cells expressing the selectablemarker, and (5) screening the cells for the expressed Ig chain.Optionally, the cells expressing the desired heavy chain or the desiredlight chain can be further selected for desirable characteristics, suchas heavy or light chain production rate or level, ability of theexpressed heavy or light chain to combine with another light or heavychain, respectively, to provide an antibody having a desired antigenbinding affinity, and/or other characteristics desirable for heavy orlight chain production or function in an antibody.

[0041] Transformed cells expressing or capable of expressing the desiredcomponent of the multimeric protein are fused by methods known in theart to form a hybrid cell expressing the multimeric protein. When themultimeric protein is an antibody, the DNA sequences encoding thedesired immunoglobulin may be composed entirely of sequences originatingfrom a single species, e.g., fully human or fully murine, or may becontain sequences originating from more than one species, e.g., ahuman-mouse chimera or CDR-grafted humanized antibody. The hybrid cellproduced antibody product may also contain a desired antigen bindingsite (variable region) linked to a desired constant region. Thus, aspecifically designed antibody may be generated with a desiredantigenicity combined with the desired isotype.

[0042] Prior art methods for independently expressing the light andheavy chains in a single host cells are known, see, for example, U.S.Pat. No. 4,816,397, European patent application publication No. 88,994,PCT published patent application WO 93/19172, U.S. Pat. No. 4,816,567,U.S. Pat. No. 4,975,369, U.S. Pat. No. 5,202,238, PCT published patentapplication WO 86/01533, PCT published patent application WO 94/02602,and European published patent application No. 273,889.

Vector Constructs

[0043] The vectors of the invention are recombinant DNA vectorsincluding, but not limited to, plasmids, phages, phagemids, cosmids,viruses, retroviruses, and the like, which insert a nucleotide sequenceinto a cell.

[0044] Methods for introducing an exogenous nucleotide sequence ofinterest into a cell, including into antibody-producing cells, are knownin the art. These methods typically include use of a DNA vector tointroduce the nucleotide sequence into the genome or a cell or cells,and then growing the cells to generate a suitable population. Nucleotidesequences may also be introduced directly into a cell by methods knownin the art.

[0045] In a preferred embodiment, nucleotide sequences are introducedinto mammalian cells according to the CaPO₄ transfer procedure describedby Graham and van der Eb (1973) Virology 52:456-467, herein specificallyincorporated by reference. Transfection of mammalian cell lines may beaccomplished by any of a number of methods known to those skilled in theart, including but not limited to, CaPO₄ precipitation, electroporation,microinjection, liposome fusion, RBC ghost fusion, protoplast fusion,and the like.

DNA Sequences

[0046] The nucleotide sequence encoding a component of the desiredmulti-component protein may be obtained as a cDNA or as a genomic DNAsequence by methods known in the art. For example, messenger RNA codingfor a desired component may be isolated from a suitable source employingstandard techniques of RNA isolation, and the use of oligo-dT cellulosechromatography to segregate the poly-A mRNA. When the productmulti-component protein is an antibody, suitable sources of desirednucleotide sequences may be isolated from mature B cells or a hybridomaculture.

[0047] In addition to the nucleotide sequence encoding the desiredcomponent of the product multi-component protein, vector constructs caninclude additional components to facilitate replication in prokaryoticand/or eukaryotic cells, integration of the construct into a eukaryoticchromosome, and markers to aid in selection of and/or screening forcells containing the construct (e.g., the detectable markers and drugresistance genes discussed above for the targeting construct). Foreukaryotic expression, the construct should preferably additionallycontain a polyadenylation sequence positioned 3′ of the gene to beexpressed. The polyadenylation signal sequence may be selected from anyof a variety of polyadenylation signal sequences known in the art.Preferably, the polyadenylation signal sequence is the SV40 earlypolyadenylation signal sequence.

Transformation of Host Cells

[0048] Antibodies have been expressed in a variety of host cells,including bacterial, yeast, and insect cells. For the production oflarge, multimeric proteins, mammalian cell expression systems generallyprovide the highest level of secreted product (Bebbington (1991)Methods: A Companion to Methods Enzymol. 2:136-145). Myeloma cells havebeen used as fusion partners for splenic cells to generate hybridomascells expressing antibodies. Transformed myeloma cells may be used asfusable host cells in the method of the invention.

Host Cells

[0049] Nonlymphoid cells lines have been investigated for use inproducing antibodies (Cattaneo & Neuberger (1987) EMBO J. 6:2753-2758;Deans et al. (1984) Proc. Natl. Acad. Sci. 81:1292-1296; Weidle et al.(1987) Gene 51:21-29). The ability of nonlymphoid cell lines to assembleand secrete fully functional antibodies may be exploited for antibodyproduction. For example, Chinese hamster ovary (CHO) cells and COS cellshave well-characterized efficient expression systems and have been usedfor both long-term and transient expression of a variety of proteins(Bebbington (1991) supra). A method for achieving a high level ofexpression of DNA sequences encoding a chimeric antibody in transformedNSO myeloma cells has been described (Bebbington et al. (1992)Bio/Technology 10:169-175).

[0050] Any mammalian cell line capable of expressing the desiredmultimeric protein and amenable to fusion is suitable for use in thepresent invention. For example, where the desired protein is anantibody, the cell line is any mammalian cell capable of expressing afunctional antibody. A preferred host cell is a mammalian myeloma cell;most preferably, an non-secreting (NS) myeloma cell (e.g., anon-secreting (NSO) myeloma). Other myeloma cells include mouse derivedP3/X63-Ag8.653, P3/NS1/1-Ag4-l(NS-1), P3/X63Ag8.U1 (P3U1), SP2/O-Ag14(Sp2/O, Sp2), PAI, F0, and BW5147; rat derived 210RCY3-Ag.2.3; and humanderived U-266AR1, GM1500-6TG-A1-2, UC729, CEM-AGR, DIR11, and CEM-T15.

Selection of Transformed Cells

[0051] Detection of transfectants with properly integrated vectorsequences can be accomplished in a number of ways, depending on thenature of the integrated sequences. If the transferred nucleotidesequence includes a selectable marker, the initial screening of thetransfected cells is to select those which express the marker. Any of avariety of selectable markers known in the art may be included in theconstruct, including dihydrofolate reductase (DHFR), guanosinephosphoryl transferase gene (gpt), neomycin resistance gene (Neo),hygromycin resistance gene (Hyg) and hypoxanthine phosphoribosyltransferase (HPRT). For example, when using a drug resistance gene,those transfectants that grow in the selection media containing the drug(which is lethal to cells that do not contain the drug resistance gene)can be identified in the initial screening. It will be appreciated thata variety of other positive, as well as negative (i.e., HSV-TK, cytosinedeaminase, and the like), selectable markers that are well known in theart can be utilized in accordance with the present invention forselection of specific cells and transfection or other events. As well, avariety of other marker genes (i.e., the LacZ reporter gene and thelike) can be utilized in similar manners.

[0052] After a period of time sufficient to allow selection to occur (inmost cases, about 2 weeks) the surviving cells are then subjected to asecond screening to identify those transfectants which express thedesired peptide component of interest. This may be accomplished by, forinstance, an immunoassay using antibodies specific for the particularimmunoglobulin class.

[0053] The protocol for the second screening depends upon the nature ofthe inserted sequences. For example, where the cell is transformed witha sequence which does not result in a secreted product, selection forthe presence of the foreign DNA can be detected by Southern blot using aportion of the exogenous sequence as a probe, or by polymerase chainreaction (PCR) using sequences derived from the exogenous sequence asamplifiers. The cells having an appropriately integrated sequence canalso be identified by detecting expression of a functional product,e.g., immuno-detection of the product. Alternatively, the expressionproduct can be detected using a bioassay to test for a particulareffector function conferred by the exogenous sequence.

[0054] Where the first host cell is transfected with DNA encoding heavychain, the expression of the heavy chain can be tested using anyconventional immunological screening method known in the art, forexample, ELISA conducted with cell lysate samples (see, for example,Colcher et al. Protein Engineering 1987 1:499-505). The cell can befurther selected for additional desirable characteristics such as heavychain production rate or level, ability of the expressed heavy chain tocombine with light chain to provide an antibody of a desired antigenbinding affinity, and other characteristics desirable for heavy chainproduction and heavy chain function in an antibody.

[0055] Nonlymphoid cells expressing a desired protein may be transfectedin a number of ways known to the art. One example of the method of theinvention is described in Example 1 below. A first CHO cell may betransfected with a vector comprising a DNA sequence encoding a desiredlight chain and a second CHO cell transfected with a vector comprising aDNA sequence encoding a desired heavy chain. Transfected cells areselected and fused. Fused cells are selected for expression of anantibody having the desired light chain Ig and heavy chain Ig.

[0056] In one embodiment, a cell expressing an Ig heavy chain gene alsoexpresses an irrelevant Ig light chain gene. In some instances,co-expression of a light chain may be required for secretion andexpression of the Ig heavy chain. Failure of a cell to secrete the heavychain peptide may make detection of transfectants more difficult sinceit necessitates assaying the cells themselves (e.g., by Northern blotanalysis or immuno-detection), as opposed to conveniently screening thecell supernatant by ELISA.

[0057] In a specific embodiment of the invention, this problem isavoided by transfecting a first host cell expressing an irrelevant lightchain with a plasmid bearing the desired heavy chain (FIG. 3). The geneencoding the irrelevant light chain may either be integrated into achromosome or be present in an episomal vector, such as bovine papillomavirus (BPV) or other episomal vector known in the art. After selectionfor transformants, expression of the heavy chain is easily confirmed byan ELISA assay of the cell lysates for secreted antibody.

[0058] Cells expressing the desired heavy chain are then fused with asecond cell that has been transfected with the desired light chain underappropriate fuseogenic conditions according to methods well known in theart (see, e.g., Harlow & Lane, supra). Any combination of cells capableof expressing a desired heavy chain or desired light chain and that canbe fused to produce a hybrid cell expressing both heavy and light chainscan be used. Thus, the first cell (e.g., expressing the desired heavychain) can be of the same or different type as the second cell (e.g.,expressing the desired light chain), e.g., the first cell can be amyeloma cell and the second cell can be a non-lymphoid cell. The fusionproduct cells which are candidates for manufacturing lines will expressthe desired heavy chain and light chain, but will have lost theirrelevant light chain. During the fusion process, random chromosomesare normally lost. Thus, it is expected that cells lacking theirrelevant Ig light chain will be generated during the fusion process.These hybrid cells can easily be identified by ELISA assay of thesupernatants for the presence of the desired chains and absence of theirrelevant chain.

[0059] Thus, in one embodiment, the desired light chain of the finalantibody product is the κ light chain. In such cases, a Igλ expressingmyeloma cell is transfected with the desired IgH gene. Aftertransfection with a plasmid carrying the desired heavy chain andselection, cells expressing the heavy chain are examined directly forexpression of the desired heavy chain, e.g., ELISA assay of thesupernatants with antibody specific to the heavy chain. The second cell,e.g., a non-secreting myeloma cell, is transfected with the κ lightchain, and transfectants detected through e.g., Northern blot analysisor immuno-detection with an antibody specific to the κ light chain. Thecells expressing the light chain can be further selected for desirablecharacteristics associated with production of a functional light chain,such as light chain production rate or level, ability of the expressedlight chain to combine with heavy chain to provide an antibody of adesired antigen binding affinity, and other characteristics desirablefor light chain production and heavy chain function in an antibody. Thecells are then fused, and the hybrid cell expressing the desired IgH/IgKantibody is selected for the presence of the κ light chain and desiredheavy chain (e.g., C_(γ)) and the absence of λ light chain, e.g., byELISA assay of the culture medium.

[0060] When the desired product antibody contains a λ light chain, thefirst cell transfected with DNA encoding the desired heavy chain willexpress a κ light chain, and final selection of hybrid cells expressingthe desired antibody will select for the presence of the λ light chainand the absence of the κ light chain.

[0061] In an alternative embodiment of the invention, selection of fusedor hybrid cells can be initially determined through the utilization ofdistinct marker genes in each of the “parental” cells or cell lines.Such technique is shown in FIG. 4. There, a parental CHO cell line, thatis DHFR, is transfected with a vector (pManu Kappa) that contains theDHFR resistance gene and the hygromycin resistance gene (HYGRO). Anotherparental CHO cell line, that is DHFR, is transfected with a vector(pManu Gamma) that contains the DHFR resistance gene and the neomycinresistance gene. Each cell line, following transfection, containsdistinct selectable markers (i.e., hygromycin resistance in the firstand neomycin/G418 resistance in the second). Thus, upon fusion,resulting “daughter” cells in which fusion has been successful will beresistant to both hygromycin and G418. The screening technique of theinvention is advantageous in that it mitigates the need to determineexpression of immunoglobulin molecules in order to determine if a fusionhas been successfully performed.

[0062] Under certain fusion conditions, cells and cell lines can becomespontaneously resistant to G418, and, possibly, other selectablemarkers. Thus, in certain embodiments of the invention, it is preferableto utilize selectable markers to which cells and cell lines are lesslikely to spontaneously generate resistance. An example of one suchmarker is the hypoxanthine phosphoribosyl transferase gene (HPRT) whichconfers resistance to hypoxanthine aminopterin. Another marker that canbe used in tandem with HPRT resistance is the LacZ gene. The LacZ geneis not a selectable marker; but, rather, acts as a marker gene which,when expressed by a cell, stains blue in the presence ofβ-galactosidase.

[0063] Thus, through following a similar scheme as described inconnection with FIG. 4, a first parental cell line, which is HPRTdeficient (such as the P3X, NSO, and NSO-bcl2 myeloma cell lines), istransfected with an antibody gene cassette. The cassette includes, forexample, appropriate antibody genes, a gene amplification system, and anHPRT selectable marker. Transfected cells can be selected through HPRTselection and cells producing high levels of antibodies can be picked. Asecond parental cell line, which is also preferably HPRT deficient, istransfected with an antibody gene cassette. The cassette includes, forexample, appropriate antibody genes, a gene amplification system, andthe LacZ gene. Transfected cells can be selected through staining withβ-gal. As will be appreciated, either the first or second parental cellline can include the light chain genes or the heavy chain genes and theother of the first or second parental cell line will contain the otherof the light or heavy chain genes. As will also be appreciated, otherselectable markers can be included in the cassettes utilized totransfect the cells. Upon fusion of the first and second parental celllines, successful fusion can be determined through HPRT selection andβ-gal staining of daughter cells. Daughter cells can be further selectedbased upon expression levels of immunoglobulin molecules.

[0064] Specific embodiments of this technique is illustrated in FIG. 5in several exemplary schemes. In the Figure, a first parental cell line,exemplified by the myeloma cell line, NSO, which is HPRT deficient, istransfected with a light chain cassette containing a gene amplificationsystem (AM), an antibody light chain gene system (V_(K)J_(K)C_(K)), andan HPRT selectable marker (HPRT) (Step 1). A second parental cell line,exemplified by any one of J558L, Ag.1, or NSO, are transfected with aheavy chain cassette containing a gene amplification system (AM), anantibody heavy chain gene system (V_(H)D_(H)J_(H)hγ), and the LacZ gene(Step 2). The transfection of J558L cell line is indicated as Step 2 a,the transfection of the Ag.1 cell line is indicated as Step 2 b, and thetransfection of the NSO cell line is indicated as Step 2 c. With respectof each Step 1 and Steps 2 a-2 c, the success of the transfection can bedetermined through the use of the selectable marker HPRT in Step 1 andthrough β-gal staining in connection with each of Steps 2 a-2 c.Additionally cells can be picked for expression of light chain (Step 1)or heavy chain (Step 2 a 2 c).

[0065] Following isolation and generation of parental cell linesincorporating the antibody gene cassettes, fusion between a parentalcell line including heavy chain genes and a parental cell line includinglight chain genes is conducted. Utilizing techniques described herein,the parental cell line resulting from Step 1 is fused with a parentalcell line resulting from Steps 2 a-2 c. This is indicated in the Figureas fusion 1-2 a, fusion 1-2 b, and fusion 1-2 c, which results in fusedcells 1-2 a, 1-2 b, and 12 c, respectively. Such fused cells can bereadily identified through dual marker selection, that is, HPRTselection and β-gal staining. Cells which have been successfully fused,will be HPRT resistant and will stain positive with β-gal.

[0066] As will be appreciated, the parental cell lines utilized infusions 1-2 a and 1-2 b additionally contain mouse mλ and rat γk genes.Thus, daughter cells from fusions 1-2 a and 1-2 b are preferablyselected to ensure that they are mλ and γK. Loss of mouse mλ genes andrat γK genes will generally occur naturally through recombination eventsduring the fusion process.

EXAMPLES

[0067] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use various constructs and perform the various methodsof the present invention and are not intended to limit the scope of whatthe inventors regard as their invention. Unless indicated otherwise,parts are parts by weight, temperature is in degrees centigrade, andpressure is at or near atmospheric pressure. Efforts have been made toensure accuracy with respect to numbers used, (e.g., length of DNAsequences, molecular weights, amounts, particular components, etc.) butsome deviations should be accounted for.

Example 1 Generation of Hybrid Cells Containing Light and Heavy IgChains

[0068] The human heavy chain Ig construct (IgH gamma) was ligated intothe pManugamma#6 vector (FIG. 4; Cell Genesys, Inc., Foster City,Calif.) containing DHFR and neo marker genes. The human kappa lightchain Ig construct was ligated into the pManukappa#14 (FIG. 4; CellGenesys, Inc.) which contains DHFR and hygromycin resistance markergenes.

Overview of the Cell Fusion Method

[0069] In general, the experiment proceeds as follows: A first cell istransfected with the pManukappa vector comprising the human kappa lightchain transgene, and MTX and hygromycin selection marker genes. A secondcell is transfected with the pManugamma vector comprising a human γ₄heavy chain transgene and Neo and MTX selectable marker genes. After theappropriate selection and amplification, the selected first and secondcells are fused to form the hybrid cell of the invention expressing ahuman antibody.

Cell Transfection

[0070] Chinese hamster ovary (CHO) cells are transfected byelectroporation as follows: DHFR-deficient CHO cells in exponentialgrowth are fed with growth medium 4 hours prior to electroporation[growth medium: DMEM/Ham's F12 (50:50 mixture; JRH BioSciences,Woodland, Calif.), 10% FBS, 2 mM glutamine, non-essential amino acids(NEAA) plus glycine, hypoxanthine and thymidine (GHT)]. Cells arecollected, washed in PBS, and resuspended in PBS to a concentration of5×10⁶ cells per 0.8 ml. The cell suspension is aliquoted into 0.4 cmelectroporation cuvettes (0.8 ml per cuvette) and 5-20 ug linearized DNAadded. The suspension is mixed and left on ice for 10 min. Each cuvetteis electroporated at 260 V and 960 uF. Each cuvette is place on ice for10 min, the cells resuspended in 20 ml growth medium, then plated onto 210 cm cell culture plates. After 48 hrs, cells from each culture plateare replated in 10 culture plates in the presence of selective media[DMEM, 4.5 g/l glucose (JRH Biosciences), 10% dialyzed FBS (LifeTechnologies, Bethesda, Md.), 5 mM glutamine, NEAA, 0.6 mg/ml G418].

Selection of Transfectants

[0071] Cells transfected with the kappa light chain transgene wereselected in the presence of methotrexate (MTX) and hygromycin. Cellswere plated 48 hr post-electroporation into 10 plates in DHFR selectivemedia [DMEM, 4.5 g/l glucose (JRH Biosciences), 10 dialyzed FBS (LifeTechnologies, Bethesda, Md.), 5 mM glutamine, NEAA, supplemented withhygromycin (Calbiochem, San Diego, Calif.) at concentrations rangingfrom 250-750 ug/ml]. Recombinant protein expression can be increased byDHFR-mediated amplification of the transfected gene. Methods forselecting cell lines bearing gene amplifications are known in the art,e.g., for example, as described in Ausubel et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York; suchmethods generally involve extended culture in medium containinggradually increasing levels of methotrexate.

[0072] Heavy chain transfectant CHO cells are selected in the presenceof MTX and neomycin following the above described procedures.

Generation of a Hybrid Cell Expressing an Antibody

[0073] Prior to fusion, PEG/DMSO fusion solution (50% PEG, 10% DMSO inPBS)(Sigma) is placed in a 37° C. incubator overnight, and 500-1000 mlincomplete Ham's F12 solution (without FCS) is filtered. At fusion, warmfusion medium and incomplete DMEM/Hams' F12 are placed in a 37° C. waterbath. A water-filled beaker and a 15 ml conical tube filled withincomplete DMEM/Ham's F12 are also placed in the water bath. Harvestedtransfected CHO cells are washed once with incomplete DMEM/Ham's F12,pelleted at 1200 rpm, resuspended in incomplete DMEM/Ham's F12, andcounted. The kappa light chain and the γ₄ transfected cells are mixed ina 1:1 ratio, and centrifuged at 800×g (2060 rpm). The following fusionsteps are followed: (1) add 1 ml PEG/DMSO fusion solution to cells over1 min period; (2) stir cells gently for 1 min; (3) add 2 ml incompleteDMEM/Ham's F12 over a 2 min period with slow stirring; and (4) add 8 mlof incomplete DMEM/Ham's F12 over a 3 min period with slow stirring. Thecells are then centrifuged at room temperature at 400×g for 5 min (1460rpm). Selection medium [complete DMEM/Ham's F12+10% FCS+250-750 ug/mlhygromycin+0.6 mg/ml G418] is added to the cell pellet. 10 ml ofselection medium are added to the cell pellet; cells are gently stirredto resuspend.

[0074] The cells are plated onto 10 cm dishes as dilutions of 1:10,1:20, and 1:40 in selection medium. The plates are refed with freshmedium every 3 days until clones appear. Clones are picked andtransferred to a 96-well plate in selection medium. As will beappreciated, growth of cells to reach confluence, which demonstratessurvival of cells through selection with hygromycin and G418, isindicative that the cells contain both the heavy and light chain Iggenes, since hygromycin resistance was contributed by the light chaingene containing parental cells and neomycin resistance was contributedby the heavy chain gene containing parental cells. As such, dual markerselection provides an expedient method to initially determine whether afusion has been successfully accomplished. Following such an initialscreen, supernatant can be assayed for expression of the desiredantibody as described below. When the wells are confluent, thesupernatant is assayed for expression of the desired antibody asdescribed below.

Selection for Desired Hybrid Cell

[0075] Expression of the desired antibody may be assayed byimmunological procedures, such as Western blot or immunoprecipitationanalysis of hybrid cell extracts, or by immunofluorescence of intactcells (using, e.g., the methods described in Ausubel et al. (1989)supra). The desired antibody is detected using antibody specific foreach component of the desired antibody, e.g., antibodies specific to thekappa light chain and γ₄ heavy chain.

Confirmation of Desired Characteristics of Antibody Produced by HybridCell

[0076] After the hybrid cell is produced and antibody production in thecell is confirmed, the hybrid cell is grown under conditions to allowexpression of the antibody and secretion of the antibody into the cellculture supernatant. For example, the cells can be grown in rollerbottles in selective growth medium (DMEM/Ham's F12 (50:50 mixture), 10%.FBS, 2 mM glutamine, non-essential amino acids plus glycine,hypoxanthine and thymidine, plus hygromycin and G418 to providecontinued selection for the heavy and light chain constructs in thehybrid cell) for several hours prior to assay. Cell culture supernatantis collected and the antibodies are tested for various desiredcharacteristics, e.g., antigen binding affinity (e.g., preferablyantigen binding affinity that is similar to that of the originalantibody from which the recombinant antibody is derived) usingimmunological assays well known in the art (e.g., ELISA, or competitionbinding assays).

[0077] The instant invention is shown and described herein in what isconsidered to be the most practical and the preferred embodiments. It isrecognized, however, that departures may be made therefrom which arewithin the scope of the invention, and that obvious modifications willoccur to one skilled in the art upon reading this disclosure.

What is claimed is:
 1. A method for producing a multi-component protein,said method comprising: (a) introducing a first nucleotide sequence intoa first cell, wherein the first nucleotide sequence encodes a firstcomponent of the multi-component protein; (b) introducing a secondnucleotide sequence into a second cell, wherein the second nucleotidesequence encodes a second component of the multi-component protein; (c)optionally, repeating step (b) for each remaining component of themulti-component protein; and (d) fusing cells produced from steps(a)-(c) to form a hybrid cell, wherein the hybrid cell expresses themulti-component protein.
 2. The method of claim 1, further comprising:(e) culturing the hybrid cells so as to express the multi-componentprotein; and (f) recovering the multi-component protein from the hybridcell culture.
 3. The method of claim 1, wherein said first cells andsaid second cells are selected from the group consisting of a mammaliancell, a myeloma cell, and a non-lymphoid cell.
 4. The multi-componentprotein of claim 1, wherein said protein is an antibody.
 5. A method forproducing an antibody, said method comprising: (a) introducing anucleotide sequence encoding a desired heavy chain into a first cell;(b) introducing a nucleotide sequence encoding a desired light chaininto a second cell; and (c) fusing the first and second cells to form ahybrid cell, wherein the hybrid cell expresses the antibody.
 6. Themethod of claim 5, further comprising: (e) culturing the hybrid cells soas to express the multi-component protein; and (f) recovering themulti-component protein from the hybrid cell culture.
 7. The method ofclaim 5, wherein said nucleotide sequence is obtained from a B-cell or ahybridoma cell, wherein said B-cell or hybridoma cell produce anantibody.
 8. The method of claim 5, wherein the first cell expresses anirrelevant light chain and expresses the desired heavy chain prior tofusion with the second cell.
 9. The method of claim 5, whereinexpression of the desired heavy chain by the first cell is determined byELISA analysis of lysate from the first cell.
 10. The method of claim 5,wherein the antibody is expressed only after fusion of said first andsecond cells.
 11. The method of claim 5, wherein the first cellexpressing the desired heavy chain is further selected for one or moredesirable characteristics.
 12. The method of claim 5, wherein both thesecond cell expressing the desired light chain and the first cellexpressing the desired heavy chain are each further selected for one ormore desirable characteristics.
 13. The method of claim 12, wherein saiddesirable characteristic is selected from the group consisting of highproduction rate of the heavy chain and high production rate of lightchain.
 14. A method for producing an antibody, said method comprising:(a) introducing a nucleotide sequence encoding a desired heavy chaininto a first cell, wherein the first cell expresses an irrelevant lightchain; (b) introducing a nucleotide sequence encoding a desired lightchain into a second cell; and (c) fusing the first and second cells toform a hybrid cell, wherein the hybrid cell expresses the antibody. 15.The method of claim 14, further comprising: (e) culturing the hybridcells so as to express the multi-component protein; and (f) recoveringthe multi-component protein from the hybrid cell culture.
 16. The methodof claim 14, wherein said irrelevant light chain is present in anepisomal vector.
 17. A multi-component protein produced by the method ofclaim
 1. 18. An antibody produced by the method of claim
 5. 19. Aantibody produced by the method of claim
 14. 20. A method for screeningfor successful fusion of a first cell containing a first nucleotidesequence encoding a desired antibody heavy chain and a second cellcontaining a second nucleotide sequence encoding a desired antibodylight chain, comprising: including a nucleotide sequence encoding afirst marker gene in the first cell; including a nucleotide sequenceencoding a second marker gene in the second cell; fusing the first celland the second cell under fuseogenic conditions to produce a fused cell;and assaying for the presence of the first and second marker genes inthe fused cell, wherein detection of the presence of the first andsecond marker genes in the fused cell indicates a successful fusion. 21.The method of claim 20, wherein the first marker gene is independentlyselected from the group consisting of the hygromycin resistance gene,the neomycin resistance gene, the hypoxanthine phosphoribosyltransferase gene, the dihydrofolate reductase gene, and the LacZreporter gene and the second marker gene is independently selected fromthe group consisting of the hygromycin resistance gene, the neomycinresistance gene, the hypoxanthine phosphoribosyl transferase gene, thedihydrofolate reductase gene, and the LacZ reporter gene.
 22. The methodof claim 20, wherein the first marker gene is the hypoxanthinephosphoribosyl transferase and the second marker gene is the LacZreporter gene.
 23. The method of claim 20, wherein the first marker geneis the LacZ reporter gene and the second marker gene is the hypoxanthinephosphoribosyl transferase gene.